Systems, devices, and methods for bodily fluid separation materials

ABSTRACT

In one embodiment described herein, a bodily fluid separation material is provided comprising a formed component capture region and a bodily fluid pass-through region. The pass-through region has structures with a reduced liquid leaching quality relative to than the capture region, wherein during separation material use, bodily fluid enters the capture region prior to entering the pass-through region. Optionally, a bodily fluid pass-through region has a reduced amount of liquid leaching material relative to than the capture region.

BACKGROUND

Certain separation materials can be used for separating plasma fromwhole blood. One example of such a separation material is an asymmetricseparation membrane, which can be used to separate solid or semi-solidcomponents from liquid components through the principle of sizeexclusion. For plasma separation, the asymmetric separation membranetraps formed blood components such as red and white blood cells in thelarger pores at the top of the separation material and allows plasma tofilter through the smaller pores at the bottom of the separationmaterial.

The filtered plasma can then be used for other purposes such as but notlimited to analyte measurement. For assay uses, plasma separationmembranes have been used commercially for large molecule assays forcertain proteins and lipids. Examples include assays directed at cardiacbiomarkers such as Troponin I, albumin, cholesterol, etc. Most of thecurrent applications of plasma separation membranes are in lateral flowassays, where the separation material is mounted on top of anotherabsorbent material which houses the assay reagents and where thereaction happens. This limits the use of this separation membrane to alimited set of assays which use mostly undiluted plasma and which can beperformed on a surface.

Unfortunately, these conventional plasma separation membranes areconfigured in manner that can negatively interfere with plasma sampleintegrity, particularly when the plasma is used for certain assays. Forexample, a hemolysis preventing agent typically used with suchseparation membranes is a substance that can leach out of the separationmembrane surface and into the collected plasma sample, which can thenresult in erroneous measurements for certain assays. Although theanti-hemolytic effect is generally desirable for sample integrity,errors associated with such coatings is not desirable.

SUMMARY

At least some disadvantages associated with the prior art are overcomeby at least some or all of the embodiments described in this disclosure.Although the embodiments herein are typically described in the contextof obtaining a blood sample, it should be understood that theembodiments herein are not limited to blood samples and can also beadapted for use with other fluid(s) or bodily sample(s).

In one embodiment described herein, it is desirable to use separationmaterials on a bodily fluid to allow for plasma-based assays. Thedesired list of assays includes not only large molecules such asproteins and lipids, but also smaller metabolites such as those that arepart of the complete metabolic panel and examples include but are notlimited to glucose, calcium, magnesium, etc . . . Since the plasmaseparation materials were not primarily designed for these assays butfor use in select types of test-strip based assays, thehemolysis-preventing agent used in these materials can interfere withother assay chemistries.

In the case of at least some bodily fluid separation materials describedherein, the separation material may have a coating of a protectivematerial such as but not limited to an anti-hemolytic material likesingle and/or double alkyl chain N-oxides of tertiary amines (NTA).Alternatively, separation material coating can constitute a combinationof an anti-hemolytic (such as surfactant, protein, sugar, or acombination of these), alongside an anti-coagulant (such as EDTA and itsderivatives or Heparin). NTA generally does not interfere with severallarge molecule assays. NTA, however, is a chelating agent that stronglybinds to di-valent cations such as calcium and magnesium ions.Unfortunately, this results in a very strong interference in certainassays used to measure, for example and not limitation, calcium andmagnesium concentrations and also for assays where Ca and Mg areco-factors for enzymes which are part of the reaction. This can resultin significant errors for such assays.

One or more of the embodiments described herein provide the benefits ofthe anti-hemolytic material but also provide a much reduced downsideeffect of the anti-hemolytic material leaching into the bodily fluid andaltering the assay results. It should be understood that the coating, insome embodiments, can be one or more of the following: anti-coagulant,anti-hemolytic, and molecules for surface coverage. Any of these mayinterfere with assays. Some embodiments disclosed herein are directedtoward multi-region separation material structures with captureregion(s) and pass-through region(s) with different surface treatments.

Optionally, these separation materials may be asymmetric ornon-asymmetric separation materials. Some embodiments have bi-layer,tri-layer, or other multi-layer configurations. Some embodiments may becontinuously asymmetric with the asymmetric region extending from anupper surface of the material to a lower surface of the material.Optionally, some embodiment may have only one or more portions of thematerial that are asymmetric while one or more other regions areisotropic in terms of pore size. Some embodiments may have an asymmetricmaterial that is then bonded to at least another material that isisotropic to create a desired pore size distribution profile. In such anembodiment, the asymmetric region may have the larger pore sizes and becoated with anti-hemolytic material. Some embodiments can haveseparation materials with gradation in coating material thickness and/orcoverage to position material such as the hemolysis-preventing materialin areas where the material is likely to be in contact with formedcomponents captured by the separation material.

By way of non-limiting example, some separation materials may be washedin a manner the preferentially removes the anti-hemolytic material fromat least one region of the separation material, such as the innerportions of the separation material, but not the exterior portions thatare more likely to come into direct contact with formed components ofthe bodily fluid. Other variations or alternative coating schemes tocreate separation materials or filter structures with areas of leachingand non-leaching materials are not excluded. Optionally, separationmaterials can also be coated with at least two different materials thatmay both leach into the bodily fluid, but at least one of thesematerials that may leach into the fluid does not impact assaymeasurements and can be used to overcoat the other material and thusdecrease the surface area exposure of the other material to the bodilyfluid.

Optionally, the separation material comprises an asymmetric porousmembrane. Optionally, the separation material is a mesh. Optionally, theseparation material comprises polyethylene (coated by ethylene vinylalcohol copolymer). Optionally, at least a portion of the separationmaterial comprises a polyethersulfone. Optionally, at least a portion ofthe separation material comprises an asymmetric polyethersulfone.Optionally, at least a portion of the separation material comprisespolyarylethersulfone. Optionally, at least a portion of the separationmaterial comprises an asymmetric polyarylethersulfone. Optionally, atleast a portion of the separation material comprises a polysulfone.Optionally, the separation material comprises an asymmetric polysulfone.Optionally, the separation material comprises a cellulose or cellulosederivative material. Optionally, the separation material comprisespolypropylene (PP). Optionally, the separation material comprisespolymethylmethacrylate (PMMA).

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.References cited herein are hereby incorporated by reference in theirentirety, except to the extent that they conflict with teachingsexplicitly set forth in this specification.

COPYRIGHT

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to facsimilereproduction of the patent documents and disclosures, as they appear inthe US Patent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever. The following notice shallapply: Copyright 2013 Theranos, Inc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side, cross-sectional view of a separation materialaccording to one embodiment described herein.

FIG. 2 shows an exploded side, cross-sectional view of separationmaterials according to one embodiment described herein.

FIG. 3 is schematic of a multi-layer separation material according toone embodiment described herein.

FIGS. 4 and 5 illustrate methods according to embodiments describedherein.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It may be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a material”may include mixtures of materials, reference to “a compound” may includemultiple compounds, and the like.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, if a device optionally contains a feature for a samplecollection unit, this means that the sample collection unit may or maynot be present, and, thus, the description includes both structureswherein a device possesses the sample collection unit and structureswherein sample collection unit is not present.

As used herein, the terms “substantial” means more than a minimal orinsignificant amount; and “substantially” means more than a minimally orinsignificantly. Thus, for example, the phrase “substantiallydifferent”, as used herein, denotes a sufficiently high degree ofdifference between two numeric values such that one of skill in the artwould consider the difference between the two values to be ofstatistical significance within the context of the characteristicmeasured by said values. Thus, the difference between two values thatare substantially different from each other is typically greater thanabout 10%, and may be greater than about 20%, preferably greater thanabout 30%, preferably greater than about 40%, preferably greater thanabout 50% as a function of the reference value or comparator value.

As used herein, a “surfactant” is a compound effective to reduce thesurface tension of a liquid, such as water. A surfactant is typically anamphiphilic compound, possessing both hydrophilic and hydrophobicproperties, and may be effective to aid in the solubilization of othercompounds. A surfactant may be, e.g., a hydrophilic surfactant, alipophilic surfactant, or other compound, or mixtures thereof. Somesurfactants comprise salts of long-chain aliphatic bases or acids, orhydrophilic moieties such as sugars. Surfactants include anionic,cationic, zwitterionic, and non-ionic compounds (where the term“non-ionic” refers to a molecule that does not ionize in solution, i.e.,is “ionically” inert). For example, surfactants useful in the reagents,assays, methods, kits, and for use in the devices and systems disclosedherein include, for example, Tergitol™ nonionic surfactants and Dowfax™anionic surfactants (Dow Chemical Company, Midland, Mich. 48642);polysorbates (polyoxyethylenesorbitans), e.g., polysorbate 20,polysorbate 80, e.g., sold as TWEEN® surfactants (ICI Americas, NewJersey, 08807); poloxamers (e.g., ethylene oxide/propylene oxide blockcopolymers) such as Pluronics® compounds (BASF, Florham Park, N.J.);polyethylene glycols and derivatives thereof, including Triton™surfactants (e.g., Triton™ X-100; Dow Chemical Company, Midland, Mich.48642) and other polyethylene glycols, including PEG-10 laurate, PEG-12laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate,PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate,PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryltrioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryllaurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate,PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castoroil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castoroil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides,polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitanlaurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearylether, tocopheryl PEG-100 succinate, PEG-24 cholesterol, polyglyceryl-10oleate, sucrose monostearate, sucrose monolaurate, sucrosemonopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenolseries, and poloxamers; polyoxyalkylene alkyl ethers such aspolyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such aspolyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fattyacid esters such as polyethylene glycol fatty acids monoesters andpolyethylene glycol fatty acids diesters; polyethylene glycol glycerolfatty acid esters; polyglycerol fatty acid esters; polyoxyalkylenesorbitan fatty acid esters such as polyethylene glycol sorbitan fattyacid esters; phosphocholines, such as n-dodecylphosphocholine, (DDPC);sodium dodecyl sulfate (SDS); n-lauryl sarcosine;n-dodecyl-N,N-dimethylamine-N-oxide (LADO); n-dodecyl-β-D-maltoside(DDM); decyl maltoside (DM), n-dodecyl-N,N-dimethylamine N-oxide (LADO);n-decyl-N,N-dimethylamine-N-oxide,1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 2-methacryloyloxyethylphosphorylcholine (MPC);1-oleoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LOPC);1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LLPG);3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS);n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;n-Decyl-N,N-dimethyl-3-ammonio-l-propanesulfonate; n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate;Tetradecanoylamidopropyl-dimethylammonio-propanesulfonate;Hexadedecanoylamidopropyl-dimethylammonio-propanesulfonate;4-n-Octylbenzoylamido-propyl-dimethylammonio Sulfobetaine; a Poly(maleicanhydride-alt-1-tetradecene), 3-(dimethylamino)-1-propylaminederivative; a nonyl phenoxylpolyethoxylethanol (NP40) surfactant;alkylammonium salts; fusidic acid salts; fatty acid derivatives of aminoacids, oligopeptides, and polypeptides; glyceride derivatives of aminoacids, oligopeptides, and polypeptides; lecithins and hydrogenatedlecithins, including lecithin, lysolecithin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid,phosphatidylserine; lysolecithins and hydrogenated lysolecithins;phospholipids and derivatives thereof; lysophospholipids and derivativesthereof, including lysophosphatidylcholine,lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidicacid, lysophosphatidylserine, PEG-phosphatidylethanolamine,PVP-phosphatidylethanolamine; carnitine fatty acid ester salts; salts ofalkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono-and di-acetylated tartaric acid esters of mono- and di-glycerides;succinylated mono- and di-glycerides; citric acid esters of mono- anddi-glycerides; lactylic esters of fatty acids, stearoyl-2-lactylate,stearoyl lactylate, succinylated monoglycerides, mono/diacetylatedtartaric acid esters of mono/diglycerides, citric acid esters ofmono/diglycerides, cholylsarcosine, caproate, caprylate, caprate,laurate, myristate, palmitate, oleate, ricinoleate, linoleate,linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate,lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, andsalts and mixtures thereof; alkylmaltosides; alkylthioglucosides; laurylmacrogolglycerides; hydrophilic transesterification products of a polyolwith at least one member of the group consisting of glycerides,vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols;polyoxyethylene sterols, derivatives, and analogues thereof;polyoxyethylated vitamins and derivatives thereof;polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof;fatty alcohols; glycerol fatty acid esters; acetylated glycerol fattyacid esters; lower alcohol fatty acids esters; propylene glycol fattyacid esters; sorbitan fatty acid esters; polyethylene glycol sorbitanfatty acid esters; sterols and sterol derivatives; polyoxyethylatedsterols and sterol derivatives; polyethylene glycol alkyl ethers; sugaresters; sugar ethers; lactic acid derivatives of mono- anddi-glycerides; hydrophobic transesterification products of a polyol withat least one member of the group consisting of glycerides, vegetableoils, hydrogenated vegetable oils, fatty acids and sterols; oil-solublevitamins/vitamin derivatives; and combinations thereof.

As used herein, the term “separator”, “separation material”, or“separation membrane” may include a mesh, a filter, a membrane, a porousmembrane, an asymmetric porous membrane, a semipermeable hollow fibermembrane, a percolating network structure, a material that can be usedfor size-exclusion of objects greater than a certain dimension, or otherfiltering material. Materials useful for the preparation of theseparator or separation material may be selected from the groupcomprising polyethylene (coated by ethylene vinyl alcohol copolymer),polyacrylates, polystyrene, polyethylene oxide, cellulose, cellulosederivatives, polyethersulfone (PES), polypropylene (PP), polysulfone(PSU), palymethylmethacrylate (PMMA), polycarbonate (PC),polyacrylonitrile (PAN), polyamide (PA), polytetrafluorethylene (PTFE),cellulose acetate (CA), regenerated cellulose, and blends or copolymersof the foregoing, or blends or copolymers with hydrophilizing polymers,including with polyvinylpyrollidone (PVP) or polyethyleneoxide (PEO).Suppliers of such materials and/or membranes include but are not limitedto BASF, Advanced Microdevices P. Ltd., International Point of CareInc., Gambro Lundia AB, Asahi Kasei Kuraray Medical Co., Ltd., GEHealthcare (Whatman division), or the like.

Referring now to FIG. 1, one embodiment of a filtering device such asbut not limited to a bodily fluid separation material 100 will now bedescribed. FIG. 1 shows a side cross-sectional view of the separationmaterial 100, showing cross-sections of the structures 102 of theseparation material. By way of non-limiting example, the separationmaterial 100 may be a size-exclusion barrier such as but not limited toa porous membrane with size-exclusion properties. Other embodiments mayuse other types of size-exclusion barrier(s). In one embodimentdescribed herein, the structures 102 are fibers in the separationmaterial with their cross-sectional views shown in FIG. 1. Optionally,the structures 102 are mesh portions of the separation material.Optionally, the structures 102 are pore walls or pore-definingstructures of the separation material. Optionally, the structures 102may be a percolating network of connected fibers, elongate members, orthe like. Some embodiments may combine one or more of the foregoing toform the separation material. Although the descriptions herein arewritten in the context of a separation material, other filter materialsor structures in sheet-like or other shapes are not excluded material.FIG. 1 shows that for the present embodiment, formed components 106 suchas but not limited to red blood cells, white blood cells, platelet, orother formed components of the bodily fluid can enter the separationmaterial 100 in a variety of directions, including from a top-downmanner, and will continue to pass through the separation material untilthe component reaches a size-constrained area where the spacing becomestoo small for the formed component 106 to proceed any further. In thisembodiment, operating under the principle of size exclusion, the formedcomponent 106 will then be constrained in the separation material 100while liquid portions and/or those components not size excluded cancontinue to pass through the separation material. In one non-limitingexample, arrows 104 show movement of formed components through theseparation material 100 of FIG. 1. Other movement, such as but notlimited to lateral, side-ways, and/or diagonal movement, is notexcluded.

Referring still to the embodiment of FIG. 1, the dotted line 120 showsthat in this embodiment, there are at least two regions 122 and 124 forthe separation material 100. It should be understood that otherembodiments can have even more regions. In this current embodiment, theregion 122 comprises a formed component capture region. In some specificembodiments as will be discussed in more detail below, it may be ananti-hemolytic, formed component capture region. By way non-limitingexample, the region 124 comprises a pass-through region that hasstructural elements spaced closely enough that formed components of thebodily fluid sample cannot completely pass through that region 124. Inat least some embodiments, the sizing and/or spacing of elements isselected such that the size-restriction technique of separation materialcomponents prevents the formed components from continuing through theseparation material. This filters out the formed components from theliquid components of the bodily fluid.

In one embodiment, because region 122 can be configured to be a formedcomponent capture region, structures in the region 122 will have morepotential direct contact with the formed components 106 and be incontact with them for a longer period of time, relative to structures inthe second region 124. Due at least in part to the greater directcontact physically and temporally, it may be desirable in it at leastsome embodiments described herein to treat the structures 102 of theregion 122 to minimize undesirable breakdown, spoilage, or otherdetrimental effect that may result from the formed components beingcaptured in the region 122. In one non-limiting example, the structures102 may be coated with an anti-hemolytic coating to prevent breakdown ofred blood cell when the bodily fluid being processed is blood. Oneembodiment of an anti-hemolytic coating may be an NTA coating.Optionally, other anti-hemolytic treatments in layer or other form mayuse material such as but not limited to n-Octyl-β-D-Glucopyranoside(OG), cell lipid bilayer intercalating material, phosphate estercontaining at least two ester linkages comprising fatty hydrocarbongroups, tri-2-ethylhexylphosphate, di-2-ethylhexylphthalate,dioctylterephthalate, anti-hemolytic surfactant(s), a surfactant such asbut not limited to polysorbate 80 mixed with any of the foregoing,and/or other anti-hemolytic material. Other anti-hemolytic material usedwith embodiments herein includes but is not limited to one or more ofthe following: anti-coagulants, proteins (such as but not limited toBSA, HSA, Heparin, Casein, etc.), surfactants (such as but not limitedto Tween, Silwet, SDS, etc.), sugars (such as but not limited tosucrose, trealose, etc.), and/or the like.

In one embodiment, the region 124 may be configured to be a liquidpass-through region positioned after the bodily fluid has passed throughregion 122. Although FIG. 1 illustrates region 124 to be next to region122, it should be understood that embodiments having intermediateregion(s) and/or space between the regions are not excluded. By way ofnon-limiting example, the pass-through region 124 may be configured nothave direct contact with the formed components. Optionally, onlystructures 108 defining part of the upper portion of the region 124 maybe in contact with any formed components 106. Optionally, onlystructures 108 defining part of the upper surface of the region 124 maybe in contact with any formed components 106. In one embodiment, theregion 124 may be have a selected structure size, spacing, and/or otherproperty that prevents formed components 106 from passing through theregion 124 so as to enable a size restriction filtering technique forremoving formed components from the bodily sample.

In at least some embodiments, because the formed components are not indirect contact with the region 124 or are only in minimal contact withregion 124, the separation material of region 124 may not be coated withthe material used in the region 122. Optionally, region 124 may beselective coated with the materials used in region 122 in a manner suchas but not limited to only those portions that might still be in contactwith formed components may be coated, which others portions of region122 are uncoated. Optionally, at least some embodiments may have some orall of region 124 coated with a material different from that of theregion 122. Optionally, at least some embodiments may have some or allof region 124 covered with the material of region 122 and then adding asecond layer of the second material over the material of region 122. Inone non-limiting example, this second material may be selected toprevent the first material leaching or otherwise entering the bodilyfluid when the liquid passes through the region 124. In at least someembodiments, the portions of region 124 covered with the material ofregion 122 is covered with the second material while other areas ofregion 124 are substantially or at least partially uncovered by eithermaterial. By way of example and not limitation, some embodiments may useHeparin and/or other anti-coagulant as the material for the secondlayer. Optionally, the material for the second layer may be a materialthat is already in the bodily fluid sample. By way of non-limitingexample, the material may be EDTA if the bodily fluid sample has alreadybeen or will be treated with EDTA. Optionally, for the second layer,some embodiments may use inert materials alone or in combination withany of the other materials listed herein.

Referring now to FIG. 2, a still further embodiment will now bedescribed. This embodiment shows a first separation material 200 and asecond separation material 210. Although only two separation materialsare shown, it should be understood that other embodiments havingadditional separation materials above, between, and/or below theseparation materials shown in FIG. 2 are not excluded. It should also beunderstood that one or more of the separation materials 200 and 210 can,within the separation materials themselves, each have additional regionstherein for different properties.

As seen in the embodiment of FIG. 2, the separation material 200functions as a capture region similar to the capture region 122 of theembodiment of FIG. 1. In the current embodiment, the separation material210 functions as a pass-through region similar to region 124 of theembodiment of FIG. 1.

Referring now to FIG. 3, this embodiment shows a tri-layer filterassembly with a first layer 300, a second layer 310, and a third layer320. For ease of illustration, the layers are shown to be similar inthickness, but configurations where all three are of differentthicknesses, or only are of different thicknesses are not excluded.Embodiments with additional layers are also not excluded. Layers canalso be formed of different materials.

It should be understood that any of the layers 300, 310, or 320 can beconfigured as a capture region, a pass-through region, or neither. Inone non-limiting example, at least the upper two layers 300 and 310 arecapture regions. They can have similar capture capabilities, oroptionally, one can be configured to be preferential capture ofcomponents while the other layer has preferential capture of componentsin a different size and/or shape regime. In another non-limitingexample, at least the upper two layers 300 and 310 are captures regions,but only one of them is coated with a material to prevent degradation ofthe formed component(s). Optionally, both of them are coated with amaterial to prevent degradation of the formed component(s). Anotherembodiment may have two layers such as layers 310 and 320 that are bothconfigured as pass-through layers. In one embodiment, neither of thelayers 310 or 320 have structures that are coated with a material toprevent degradation of the formed component(s). Optionally, at least oneof the layers 310 or 320 has structures that are coated with a materialto prevent degradation of the formed component(s). Optionally, someembodiments have both of the layers 310 or 320 have structures that arecoated with a material to prevent degradation of the formedcomponent(s).

SEPARATION MATERIAL TREATMENT

By way of example and not limitation, in order to be able to useseparation materials for producing plasma suitable for a greater rangeof assays, several separation material treatment methods have beenidentified. Some of these techniques may involve treatment of separationmaterials after they are formed. Some of the techniques may involveforming the separation materials in a way that does not involveadditional treatment after separation material formation. Optionally,some techniques may use both separation material formation andpost-formation treatment to create a desired configuration.

1. Separation material wash: In one embodiment described herein, bycontrolled washing of the coated plasma separation material by waterand/or buffer solutions, most of the hemolysis-preventing agents can beremoved. FIG. 4 shows that a washing mechanism, such as but not limitedto a nozzle 400 directing washing fluid (as indicated by the arrows)towards the target separation material 402, can be used to reduce atleast some of the coating off of the separation material. This cancreate a preferential change in the amount of coating in selected areasof the separation material. One example may show removal or at leastreduction of coating on one side of the separation material. Optionally,some may direct the wash fluid to wash coating off of an interior regionof the separation material. Other configurations where portions ofcoating are removed from other select areas are not excluded.

In one embodiment described herein, a carefully controlled washing isdesirable so as to not completely remove the hemolysis preventingagent—which would result in hemolysis. In contrast, insufficient washwill result in sufficient amount of the hemolysis preventing agentleaching into the plasma and causing hemolysis. Thus, in onenon-limiting example, a reduced amount of coating, or coating ininterior portions of the separation material can be acceptable.Optionally, as seen in FIG. 5, some embodiments may also use a bath 410of wash fluid that preferentially removes coating material from certainareas of the separation material. Optionally, spray washing and bathsoaking, or vice versa, may be combined for use on a separationmaterial. This processing may occur sequentially or simultaneously.

2. Custom separation material coating: In another embodiment describedherein, both coated and uncoated versions of the plasma separationmaterial can be coated using a custom formulation which is compatiblewith assay chemistries. The coating may contain one or more of thefollowing: proteins, surfactants, sugars, organic and inorganic salts,anti-coagulants, etc. In one non-limiting example, the coating could beapplied to an initially un-coated separation material to preventhemolysis. Optionally, an initially coated separation material may befurther coated to prevent assay interfering substances from leachinginto the bodily fluid from the separation material.

3. Charge Neutralization: In one embodiment described herein, separationmaterial surface charge can be neutralized to prevent retention ofsmall, oppositely charged ions. For example, the separation materialwith NTA coating has a negatively-charged surface, which can beneutralized to prevent retention of positively charged Ca++ ions.Optionally, if a coating has a positively-charged surface and is in turnattracting negatively charged ions in a detrimental manner, the memberwill be treated to neutralize the undesired charge condition.

4. Other techniques and/or materials may also be used to create a filtersuch as a separation material that has anti-hemolytic qualities on thecapture surfaces of the filter and non-leaching qualities on othersurfaces of the filter. Some embodiments may combine one or more of theforegoing techniques on a separation material. By way of non-limitingexample, one embodiment may have coated and uncoated regions on aseparation material along with having been treated to achieve chargeneutralization before, during, and/or after coating.

EXAMPLES

Using a dynamic wash technique, asymmetric membranes were washed withhigh performance liquid chromatography (HPLC) grade water and thentested. In one non-limiting example, the membrane has a pore volume of 2μL per 10 mm² of membrane. The pore loading is defined as the ratio ofthe total volume of blood to the pore volume. For a blood volume of 40μL with membrane surface area of 100 mm², this corresponds to a poreloading of 2X. The wash procedure comprised pre-mounting membrane in afixture for filtration. In this particular example, about 600 uL ofwater is directed through the membrane and then the water is discarded.This wash process of directing water through the membrane was repeated,which in this particular example, involved repeating the wash five (5)times. After washing, the membranes are allowed to dry. Filtration ofthe dynamically washed membranes were then tested.

Washing by way of soaking (“static wash”) rather than the flow-throughtechnique (“dynamic wash”) can create differences in the performance ofthe resulting membrane. In at least some static washed membranes,anti-hemolytic is preferentially removed from the large pore region. Inat least some dynamic wash membranes, anti-hemolytic is preferentiallyremoved from the small pore region. This asymmetry in coating materialmay be desirable when the formed blood components contact the membranewhere the pores are larger while only plasma contacts the smallestpores. Hemolysis prevention happens only in the regions where RBCs canenter or be contacted (i.e. the large pore region). It is not possibleto hemolyze plasma and thus coating the small pore region withanti-hemolytic does not result in noticeable performance benefit. Asnoted herein, the excess anti-hemolytic may have adverse impact on assayresults for the assays sensitive to excess anti-hemolytic coating.

In static wash, diffusion dominates removal of anti-hemolytic. In someembodiments of the membrane, large pores may be ˜50X bigger than smallpores. Mass diffusion rate is proportional to cross sectional flow area.Thus diffusion rate of anti-hemolytic away from membrane on large poreside may be ˜2500X greater than on small pore side. Thus, without beingbound to any particular theory, total removal should be much greater onlarge pore side, where the RBCs contact the membrane.

In dynamic wash, shear dominates removal of anti-hemolytic. Shearincreases dramatically with decreasing diameter. Without being bound toany particular theory, total removal should be greater in small poreregions, where shear is most significant.

In yet another embodiment, the coating on the membrane can be a materialthat provides a negative charge. Without being bound to any particulartheory, a negative charge repels formed blood component that have anegative polarity, and thereby reduces mechanical trauma inflicted onsuch formed blood components via contact with the membrane duringfiltration. Some embodiments may use formulations with negativelycharged substances to coat all or optionally selective areas on themembrane. One embodiment may use casein 0.5%, Tween 20 1.35%, sucrose5%, 15 minute soak time. Optionally, one embodiment may use Li-Heparin50 mg/mL, sucrose 5%. Optionally, one embodiment may use Li-Heparin 50mg/mL, Tween 80 1.35%, sucrose 5%. Optionally, one embodiment may useCasein 1.0%, Tween 20 2.70%, sucrose 5%. Optionally, one embodiment mayuse Li-Heparin 100 mg/mL, Tween 20 2.70%, sucrose 5%.

While the teachings has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, with any of the above embodiments, it should be understoodthat the fluid sample may be whole blood, diluted blood, interstitialfluid, sample collected directly from the patient, sample that is on asurface, sample after some pre-treatment, or the like. Although theembodiments herein are described in the context of an anti-hemolyticcoating, it should be understood that these embodiments may also beconfigured for use with other types of coatings, including but notlimited to other coatings which may undesirably mix into the bodilyfluid upon prolonged fluid exposure. Other material used withembodiments herein may include but is not limited to one or more of thefollowing: anti-coagulants, proteins (BSA, HSA, Heparin, Casein, etc.),surfactants (Tween, Silwet, SDS, etc.), sugars (sucrose, trealose, etc.)

Although the embodiments herein are described in the context ofcapturing formed components such as blood cells or platelets, it shouldbe understood that these embodiments can also be adapted for use withfluid containing other solid, semi-solid, or formed components orparticles. Although the embodiments herein are described in the contextof separation material, it should be understood that these embodimentscan also be adapted for use other filter materials such as meshes,porous layers, or other layer like materials or structures.

Additionally, concentrations, amounts, and other numerical data may bepresented herein in a range format. It is to be understood that suchrange format is used merely for convenience and brevity and should beinterpreted flexibly to include not only the numerical values explicitlyrecited as the limits of the range, but also to include all theindividual numerical values or sub-ranges encompassed within that rangeas if each numerical value and sub-range is explicitly recited. Forexample, a size range of about 1 nm to about 200 nm should beinterpreted to include not only the explicitly recited limits of about 1nm and about 200 nm, but also to include individual sizes such as 2 nm,3 nm, 4 nm, and sub-ranges such as 10 nm to 50 nm, 20 nm to 100 nm, etc. . . .

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications mentioned herein are incorporated herein by referenceto disclose and describe the structures and/or methods in connectionwith which the publications are cited. The following applications arefully incorporated herein by reference for all purposes:

OTHER EMBODIMENTS

In one embodiment described herein, a bodily fluid separation materialis provided comprising a formed component capture region and a bodilyfluid pass-through region. The pass-through region has structures with areduced liquid leaching quality relative to than the capture region,wherein during separation material use, bodily fluid enters the captureregion prior to entering the pass-through region. Optionally, a bodilyfluid pass-through region has a reduced amount of liquid leachingmaterial relative to than the capture region.

In another embodiment described herein, a bodily fluid separationmaterial is provided comprising an anti-hemolytic and formed componentcapture region; and a bodily fluid pass-through region having lessanti-hemolytic material than the capture region, wherein duringseparation material use, bodily fluid enters the capture region prior toentering the pass-through region.

In yet embodiment described herein, a bodily fluid separation materialis provided comprising a first filter region of the separation materialhaving an anti-hemolytic coating and mesh spacing sized to constrainformed blood components therein; a second filter region of theseparation material having mesh spacing smaller than mesh spacing of thefirst filter region and configured to have an amount of anti-hemolyticcoating less than that of the first region.

In a still further embodiment described herein, a bodily fluidseparation material is provided comprising a percolating network ofstructures wherein a first region of the percolating network with ananti-hemolytic coating on structures in the region, said structuressized and spaced to allow formed blood components to enter the firstregion but constraining blood components therein from passing completelythrough the first region; and a second region of the percolating networkwith a reduced anti-hemolytic coating on structures sized and spaced toprevent formed blood components from entering the second region, whereinbodily fluid passes through the first region prior to reaching thesecond region.

It should be understood that embodiments herein may be adapted toinclude one or more of the following features. For example, theseparation material may be an asymmetric separation material.Optionally, the anti-hemolytic material on the separation materialcomprises single and/or double alkyl chain N-oxides of tertiary amines(NTA). Optionally, the first region comprises a first separationmaterial layer and the second region comprises a second separationmaterial layer. Optionally, the separation material comprises a firstseparation material coupled to a second separation material. Optionally,the separation material comprises at least two separate separationmaterials. Optionally, there may be at least another region of theseparation material between the first region and the second region.Optionally, the first region of the separation material may be in fluidcommunication with the second region. Optionally, the first region maybe spaced apart from the second region.

In yet another embodiment described herein, a method is provided forforming a bodily fluid separation material. The method comprises coatingthe separation material with an anti-hemolytic coating on a first regionand a second region of the separation material; reducing anti-hemolyticeffect of the second region of the separation material relative to thefirst region, wherein when the separation material is in operation,bodily fluid passes through the first region prior to reaching thesecond region.

It should be understood that embodiments herein may be adapted toinclude one or more of the following features. For example, the methodmay include reducing the anti-hemolytic effect by washing off at least aportion of the anti-hemolytic coating on the second region. Optionally,washing off comprises directing solvent through the separation material.Optionally, washing off comprises soaking only a portion of theseparation material in a solvent. Optionally, reducing theanti-hemolytic effect comprises adding another coating of a differentmaterial over the anti-hemolytic coating on the second region.Optionally, reducing the anti-hemolytic effect comprises treating theseparation material to bring its electrical charge state to a neutralstate and thus reduce the attraction of ions that increase theanti-hemolytic effect.

In yet another embodiment described herein, a method is provided forforming a bodily fluid separation material. The method comprises coatingat least a first region of the separation material with ananti-hemolytic coating; not coating at least second region of theseparation material with the anti-hemolytic coating. Optionally, someembodiments have a bilayer structure based on a substantially evencoating of anti-hemolytic material, but instead has a region ofsubstantially greater pore size than another region. Although thematerial may be asymmetric, it is a not a linear gradient, but insteadhas a rapid change in pore size at an inflection point when pore size isgraphed in depth from top of the layer to bottom of the layer.

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications mentioned herein are incorporated herein by referenceto disclose and describe the structures and/or methods in connectionwith which the publications are cited. The following applications arefully incorporated herein by reference for all purposes: U.S. Pat. Nos.8,088,593; 8,380,541; U.S. patent application Ser. No. 13/769,798, filedFeb. 18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb.18, 2013; U.S. patent application Ser. No. 61/766,113 filed Feb. 18,2013, U.S. patent application Ser. No. 13/244,947 filed Sep. 26, 2011;PCT/US2012/57155, filed Sep. 25, 2012; U.S. application Ser. No.13/244,946, filed Sep. 26, 2011; U.S. patent application Ser. No.13/244,949, filed Sep. 26, 2011; and U.S. application Ser. No.61/673,245, filed Sep. 26, 2011, U.S. patent application Ser. No.61/786,351 filed Mar. 15, 2013, U.S. patent application Ser. No.61/697,797 filed Sep. 6, 2012, U.S. patent application Ser. No.61/799,221 filed Mar. 15, 2013, and U.S. patent application Ser. No.61/733,886 filed Dec. 5, 2012, the disclosures of which patents andpatent applications are all hereby incorporated by reference in theirentireties for all purposes.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. Any feature, whetherpreferred or not, may be combined with any other feature, whetherpreferred or not. The appended claims are not to be interpreted asincluding means-plus-function limitations, unless such a limitation isexplicitly recited in a given claim using the phrase “means for.” Itshould be understood that as used in the description herein andthroughout the claims that follow, the meaning of “a,” “an,” and “the”includes plural reference unless the context clearly dictates otherwise.For example, a reference to “an assay” may refer to a single assay ormultiple assays. Also, as used in the description herein and throughoutthe claims that follow, the meaning of “in” includes “in” and “on”unless the context clearly dictates otherwise. Finally, as used in thedescription herein and throughout the claims that follow, the meaning of“or” includes both the conjunctive and disjunctive unless the contextexpressly dictates otherwise. Thus, the term “or” includes “and/or”unless the context expressly dictates otherwise.

1-25. (canceled)
 26. A filter device comprising: a bodily fluidseparation material comprising: 1) a first capture region formed of afirst material and having an anti-hemolytic surface coating over thefirst material; 2) a second capture region formed of the first materialand without the anti-hemolytic surface coating over the first material;and 3) a bodily fluid pass-through region formed of the first materialand comprising pass-through openings sized so that formed components ofthe bodily fluid cannot enter said openings in the bodily fluidpass-through region, wherein the second capture region is between thefirst capture region and the bodily fluid pass-through region.
 27. Thedevice of claim 26 wherein at least a portion of the separation materialcomprises an asymmetric polyethersulfone.
 28. The device of claim 26wherein at least a portion of the separation material comprisespolyarylethersulfone.
 29. The device of claim 26 wherein at least aportion of the separation material comprises an asymmetricpolyarylethersulfone.
 30. The device of claim 26 wherein at least aportion of the separation material comprises a polysulfone.
 31. Thedevice of claim 26 wherein the separation material comprises anasymmetric polysulfone.
 32. The device of claim 26 wherein theanti-hemolytic surface coating comprises single and/or double alkylchain N-oxides of tertiary amines (NTA).
 33. A filter device comprising:a bodily fluid separation material comprising: 1) a first capture regionformed of a first material and having an anti-hemolytic surface coatingover the first material; 2) a second capture region formed of the firstmaterial and without the anti-hemolytic surface coating over the firstmaterial; and 3) a bodily fluid pass-through region formed of the firstmaterial and comprising pass-through openings sized so that formedcomponents of the bodily fluid cannot enter said openings in the bodilyfluid pass-through region, wherein the second capture region is betweenthe first capture region and the bodily fluid pass-through region;wherein the first capture region defines a first filter region of theseparation material having an anti-hemolytic coating and pore spacingsized to constrain formed blood components therein; wherein the bodilyfluid pass-through region defines a second filter region of theseparation material having pore spacing smaller than pore spacing of thefirst filter region with pores sized so that formed components do notenter the second filter region and configured to have an amount ofanti-hemolytic coating less than that of the first region.
 34. Thedevice of claim 33 wherein the separation material comprises at leasttwo separate separation materials.
 35. The device of claim 33 furthercomprising at least another region of the separation material betweenthe first region and the second region.
 36. The device of claim 33 wherethe first region is in fluid communication with the second region. 37.The device of claim 33 wherein the first region is spaced apart from thesecond region.
 38. The device of claim 25 wherein the first captureregion, second capture region, and the bodily fluid pass-through regioncomprise a percolating network.
 39. The device of claim 26 wherein theseparation material has a flat, planar shape.
 40. A kit comprising thedevice of claim 26 wherein at least a portion of the separation materialcomprises a polyethersulfone.
 41. A filter device comprising: a bodilyfluid separation material comprising: 1) a first capture region formedof a first material and having an anti-hemolytic surface coating overthe first material; 2) a second capture region formed of the firstmaterial and without the anti-hemolytic surface coating over the firstmaterial; and 3) a bodily fluid pass-through region formed of the firstmaterial and comprising pass-through openings sized so that formedcomponents of the bodily fluid cannot enter said openings in the bodilyfluid pass-through region, wherein the second capture region is betweenthe first capture region and the bodily fluid pass-through region;wherein the first material comprises an asymmetric polysulfone.